Albumin-free factor VIII formulations

ABSTRACT

An albumin-free Factor VIII formulation comprising: 4% to 10% of a bulking agent selected from the group consisting of mannitol, glycine and alanine; 1% to 4% of a stabilizing agent selected from the group consisting of sucrose, trehalose, raffinose, and arginine; 1 mM to 5 mM calcium salt; 100 mM to 300 mM NaCl; and a buffering agent. Alternatively, the formulation can comprise 2% to 6% hydroxyethyl starch; 1% to 4% of a stabilizing agent selected from the group consisting of sucrose, trehalose, raffinose, and arginine; 1 mM to 5 mM calcium salt; 100 mM to 300 mM NaCl; and a buffering agent. In a further embodiment, the formulation can comprise: 300 mM to 500 mM NaCl; 1% to 4% of a stabilizing agent selected from the group consisting of sucrose, trehalose, raffinose, and arginine; 1 mM to 5 mM calcium salt; and a buffering agent.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/764,770 filed Jun. 18, 2007 (now U.S. Pat. No. 8,058,226 issued Nov.15, 2011), which is a continuation of U.S. application Ser. No.11/434,634 filed May 15, 2006 (now U.S. Pat. No. 7,247,707 issued Jul.24, 2007), which is a divisional of U.S. application Ser. No. 10/610,723filed Jul. 1, 2003 now U.S. Pat. No. 7,087,723 issued Aug. 8, 2006),which is a continuation of U.S. application Ser. No. 09/507,011 filedFeb. 22, 2000 (now U.S. Pat. No. 6,586,573 issued Jul. 1, 2003), whichis a continuation-in-part of U.S. application Ser. No. 09/452,752 filedDec. 1, 1999 now abandoned), which is a continuation-in-part of U.S.application Ser. No. 09/255,279 filed Feb. 22, 1999 (now abandoned).

BACKGROUND OF THE INVENTION

Factor VIII is a protein found in blood plasma which acts as a cofactorin the cascade of reactions leading to blood coagulation. A deficiencyin the amount of Factor VIII activity in the blood results in theclotting disorder known as hemophilia A, an inherited conditionprimarily affecting males. Hemophilia A is currently treated withtherapeutic preparations of Factor VIII derived from human plasma ormanufactured using recombinant DNA technology. Such preparations areadministered either in response to a bleeding episode (on-demandtherapy) or at frequent, regular intervals to prevent uncontrolledbleeding (prophylaxis).

Factor VIII is known to be relatively unstable in therapeuticpreparations. In blood plasma, Factor VIII is usually complexed withanother plasma protein, von Willebrand factor (vWF), which is present inplasma in a large molar excess to Factor VIII and is believed to protectFactor VIII from premature degradation. Another circulating plasmaprotein, albumin, may also play a role in stabilizing Factor VIII invivo. Currently marketed Factor VIII preparations therefore primarilyrely on the use of albumin and/or vWF to stabilize Factor VIII duringthe manufacturing process and during storage.

The albumin and vWF used in currently marketed Factor VIII preparationsis derived from human blood plasma, however, and the use of suchmaterial has certain drawbacks. Because a large molar excess of albumincompared to Factor VIII is generally added in order to increase thestability of the Factor VIII in such preparations, it is difficult tocharacterize the Factor VIII protein itself in these preparations. Theaddition of human-derived albumin to Factor VIII is also perceived asbeing a disadvantage with respect to recombinantly-produced Factor VIIIpreparations. This is because recombinantly-derived Factor VIIIpreparations, in the absence of such added albumin, would otherwisecontain no human-derived proteins, and the theoretical risk oftransmitting a virus would be reduced.

Several attempts to formulate Factor VIII without albumin or vWF (orwith relatively low levels of these excipients) have been described. Forexample, U.S. Pat. No. 5,565,427 (EP 508 194) to Freudenberg (assignedto Behringwerke) describes Factor VIII preparations which containparticular combinations of detergent and amino acids, specificallyarginine and glycine, in addition to excipients such as sodium chlorideand sucrose. The detergent, polysorbate 20 or polysorbate 80, isdescribed as being present in amounts of between 0.001 to 0.5% (v/v),while arginine and glycine are present in amounts of between 0.01 to 1mol/l. Sucrose is described as being present in amounts of between 0.1and 10%. Example 2 of this patent asserts that solutions of (1) 0.75%sucrose, 0.4 M glycine, and 0.15M NaCl, and (2) 0.01 M sodium citrate,0.08 M glycine, 0.016M lysine, 0.0025 M calcium chloride, and 0.4 Msodium chloride were not stable in solution over 16 hours, whereassolutions of (3) 1% sucrose, 0.14 M arginine, 0.1 M sodium chloride and(4) 1% sucrose, 0.4 M glycine, 0.14 M arginine, 0.1 M sodium chloride,and 0.05% Tween 80 exhibited stability.

U.S. Pat. No. 5,763,401 (EP 818 204) to Nayer (assigned to Bayer) alsodescribes a therapeutic Factor VIII formulation without albumin,comprising 15-60 mM sucrose, up to 50 mM NaCl, up to 5 mM calciumchloride, 65-400 mM glycine, and up to 50 mM histidine. The followingspecific formulations were identified as being stable: (1) 150 mM NaCl,2.5 mM calcium chloride, and 165 mM mannitol; and (2) 1% sucrose, 30 mMsodium chloride, 2.5 mM calcium chloride, 20 mM histidine, and 290 mMglycine. A formulation containing higher amounts of sugar (10% maltose,50 mM NaCl, 2.5 mM calcium chloride, and 5 mM histidine) was found toexhibit poor stability in the lyophilized state compared withformulation (2).

U.S. Pat. No. 5,733,873 (EP 627 924) to Osterberg (assigned to Pharmacia& Upjohn) discloses formulations which include between 0.01-1 mg/ml of asurfactant. This patent discloses formulations having the followingranges of excipients: polysorbate 20 or 80 in an amount of at least 0.01mg/ml, preferably 0.02-1.0 mg/ml; at least 0.1 M NaCl; at least 0.5 mMcalcium salt; and at least 1 mM histidine. More particularly, thefollowing specific formulations are disclosed: (1) 14.7-50-65 mMhistidine, 0.31-0.6 M NaCl, 4 mM calcium chloride, 0.001-0.02-0.025%polysorbate 80, with or without 0.1% PEG 4000 or 19.9 mM sucrose; and(2) 20 mg/ml mannitol, 2.67 mg/ml histidine, 18 mg/ml NaCl, 3.7 mMcalcium chloride, and 0.23 mg/ml polysorbate 80.

Other attempts to use low or high concentrations of sodium chloride havealso been described. U.S. Pat. No. 4,877,608 (EP 315 968) to Lee(assigned to Rhone-Poulenc Rorer) teaches formulations with relativelylow concentrations of sodium chloride, namely formulations comprising0.5 mM-15 mM NaCl, 5 mM calcium chloride, 0.2 mM-5 mM histidine, 0.01-10mM lysine hydrochloride and up to 10% sugar. The “sugar” can be up to10% maltose, 10% sucrose, or 5% mannitol.

U.S. Pat. No. 5,605,884 (EP 0 314 095) to Lee (assigned to Rhone-PoulencRorer) teaches the use of formulations with relatively highconcentrations of sodium chloride. These formulations include 0.35 M-1.2M NaCl, 1.5-40 mM calcium chloride, 1 mM-50 mM histidine, and up to 10%of a “sugar” such as mannitol, sucrose, or maltose. A formulationcomprising 0.45 M NaCl, 2.3 mM calcium chloride, and 1.4 mM histidine isexemplified.

International Patent Application WO 96/22107 to Roser (assigned toQuadrant Holdings Cambridge Limited) describes formulations whichinclude the sugar trehalose. These formulations comprise: (1) 0.1 MNaCl, 15 mM calcium chloride, 15 mM histidine, and 1.27 M (48%)trehalose; or (2) 0.011% calcium chloride, 0.12% histidine, 0.002% Tris,0.002% Tween 80, 0.004% PEG 3350, 7.5% trehalose, and either 0.13% or1.03% NaCl.

Other therapeutic Factor VIII formulations of the prior art generallyinclude albumin and/or vWF for the purpose of stabilizing Factor VIIIand are therefore not relevant to the present invention. For example,U.S. Pat. No. 5,328,694 (EP 511 234) to Schwinn (assigned to OctapharmaAG) describes a formulation which includes 100-650 mM disaccharide and100 mM-1.0 M amino acid. Specifically, the following formulations aredisclosed: (1) 0.9 M sucrose, 0.25 M glycine, 0.25 M lysine, and 3 mMcalcium chloride; and (2) 0.7 M sucrose, 0.5 M glycine, and 5 mM calciumchloride.

While several attempts have been made to formulate Factor VIII withoutalbumin or vWF, there remains a need for therapeutic Factor VIIIformulations which are stable in the absence of albumin or otherproteins.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to therapeutic Factor VIII compositionswhich are stable in the absence of albumin. In particular, the presentinvention comprises a Factor VIII composition comprising, in addition toFactor VIII: 4% to 10% of a bulking agent selected from the groupconsisting of mannitol, glycine and alanine; 1% to 4% of a stabilizingagent selected from the group consisting of sucrose, trehalose,raffinose, arginine; 1 mM to 5 mM calcium salt; 100 mM to 300 mM NaCl;and a buffering agent for maintaining a pH of approximately between 6and 8. This composition can additionally comprise a surfactant such aspolysorbate 20, polysorbate 80, Pluronic F68, or Brij 35. When thesurfactant is polysorbate 80, it should be present in an amount of lessthan 0.1%.

The buffer of the Factor VIII compositions according to the presentinvention is preferably present in a concentration of from 10 mM to 50mM, and is preferably selected from the group consisting of histidine,Tris, BIS-Tris Propane, PIPES, MOPS, HEPES, MES and ACES.Advantageously, the buffering agent is either histidine or Tris. TheFactor VIII composition of the present invention can further comprise anantioxidant.

The Factor VIII compositions of the present invention include both abulking agent and a stabilizer. The bulking agent can be present in anamount of from about 6% to about 8%, preferably about 8%. Thestabilizing agent is preferably present in an amount of about 2%. Sodiumchloride is also present in these compositions, preferably in an amountof from 150 to 350 mM, and more preferably in an amount of about 225 mM.The calcium salt of the composition is also preferably calcium chloride,and the composition itself is preferably in lyophilized form.

In another embodiment, the present invention can comprise a Factor VIIIcomposition formulated without adding albumin which includes thefollowing excipients in addition to Factor VIII: 2% to 6% hydroxyethylstarch; 1% to 4% of a stabilizing agent selected from the groupconsisting of sucrose, trehalose, raffinose, arginine; 1 mM to 5 mMcalcium salt; 100 mM to 300 mM NaCl; and a buffering agent formaintaining a pH of approximately between 6 and 8. Preferably, such acomposition comprises about 4% hydroxyethyl starch, and the NaCl ispresent in an amount of 200 mM. The stabilizing agent is also preferablypresent in an amount of about 2%.

In a further embodiment, the present invention includes a Factor VIIIcomposition, formulated without albumin, comprising: 300 mM to 500 mMNaCl; 1% to 4% of a stabilizing agent selected from the group consistingof sucrose, trehalose, raffinose, arginine; 1 mM to 5 mM calcium salt;and a buffering agent for maintaining a pH of approximately between 6and 8. Preferably, the NaCl is present in a concentration of about 400mM.

In yet another embodiment, the present invention comprises a process forlyophilizing an aqueous Factor VIII composition in a container using alyophilizer, wherein the process comprises an initial freezing step, andthe initial freezing step further comprises the steps of: (a) loweringthe temperature of the lyophilizer chamber to at least about −45° C.;(b) raising the temperature of the chamber to between about −15° C. and−25° C.; and subsequently (c) lowering the temperature of the chamber toat least about −45° C. In this process, the temperature of the chamberis preferably lowered or raised at a rate of between about 0.5° C. andabout 1.0° C. per minute. In step (a), the temperature is preferablymaintained for about 1 hour, and is lowered to about −55° C. In step (b)the temperature is preferably maintained be −15° C. and −25° C. forbetween 1 and 3 hours, and more preferably is at −22° C., and thetemperature in step (c) is preferably maintained for about 1 hour. TheFactor VIII composition used in this process preferably comprisesbetween 4% and 10% of an agent selected from the group consisting ofmannitol, glycine and alanine, and also preferably comprises between 1%and 4% of an agent selected from the group consisting of sucrose,trehalose, raffinose, and arginine. In addition, the Factor VIIIcomposition used in this process also preferably comprises between 100mM and 300 mM NaCl.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the terms below and variations thereof shall be definedas follows, unless otherwise indicated:

Factor VIII—The Factor VIII molecule exists naturally and in therapeuticpreparations as a heterogeneous distribution of polypeptides arisingfrom a single gene product (see, e.g., Andersson et al., Proc. Natl.Acad. Sci. USA, 83, 2979-2983, May 1986). The term “Factor VIII” as usedherein refers to all such polypeptides, whether derived from bloodplasma or produced through the use of recombinant DNA techniques.Commercially available examples of therapeutic preparations containingFactor VIII include those sold under the trade names of HEMOFIL M andRECOMBINATE (available from Baxter Healthcare Corporation, Deerfield,Ill., U.S.A.). Other preparations currently in development compriseprimarily a single subpopulation of Factor VIII molecules which lack theB domain portion of the molecule.

International Unit, IU—International Unit, or IU, is a unit ofmeasurement of the blood coagulation activity (potency) of Factor VIIIas measured by a standard assay, such as one of the following:

One stage assay. One stage assays are known to the art, such as thatdescribed in Lee, Martin L, et al., An Effect of Predilution on PotencyAssays of Factor VIII Concentrates, Thrombosis Research (Pergamon PressLtd.) 30, 511-519 (1983).

Chromogenic assay. Chromogenic assays may be purchased commercially,such as the Coatest Factor VIII, available from Chromogenix AB, Molndal,Sweden.

Anneal—The term anneal shall be used to indicate a step in thelyophilization process of a pharmaceutical preparation undergoinglyophilization, prior to the freeze-drying of the preparation, in whichthe temperature of the preparation is raised from a lower temperature toa higher temperature and then cooled again after a period of time.

Bulking Agent—For the purposes of this application, bulking agents arethose chemical entities which provide structure to the “cake” orresidual solid mass of a pharmaceutical preparation after it has beenlyophilized and which protect it against collapse. A crystallizablebulking agent shall mean a bulking agent as described herein which canbe crystallized during lyophilization, other than sodium chloride. HESis not included in this group of crystallizable bulking agents.

Freeze-drying, freezing, lyophilizing—“Freeze-drying,” unless otherwiseindicated by the context in which it appears, shall be used to denotethe portion of a lyophilization process in which the temperature of apharmaceutical preparation is raised in order to drive water out of thepreparation. The “freezing” steps of a lyophilization process are thosesteps which occur prior to the freeze-drying stage. “Lyophilizing,”unless otherwise indicated, shall refer to the entire process oflyophilization, including both the freezing steps and the freeze-dryingsteps.

Unless otherwise noted, percentage terms express weight/volumepercentages and temperatures are in the Celsius scale.

Formulation Components

The Factor VIII compositions of the present invention include bulkingagents, stabilizing agents, buffering agents, sodium chloride, calciumsalts, and, advantageously, other excipients. These excipients have beenchosen in order to maximize the stability of Factor VIII in lyophilizedpreparations. However, the Factor VIII compositions of the presentinvention exhibit stability in the liquid state as well.

The bulking agents used in the present formulations, which form thecrystalline portion of the lyophilized product (except in the case ofHES), are selected from the group consisting of mannitol, glycine,alanine, and hydroxyethyl starch (HES). Mannitol, glycine, or alanineare present in an amount of 4-10%, preferably 6-9%, and more preferablyabout 8%. When HES is used as a bulking agent, it is present in anamount of 2-6%, preferably 3-5%, and more preferably about 4%.

The stabilizing agents used in the formulations of the present inventionare selected from the group consisting of sucrose, trehalose, raffinose,and arginine. These agents are present in the formulations of thepresent invention in an amount of between 1-4%, preferably 2-3%, morepreferably about 2%. Sorbitol and glycerol were evaluated as possiblestabilizers but were found to be poor stabilizers in the presentformulations.

Sodium chloride is included in the present formulations in an amount of100-300 mM, preferably 150-250 mM, and most preferably about 225 mM. Inone embodiment of the present invention, sodium chloride itself can beused without any of the aforementioned bulking agents, in which case itwould be included in the formulation in an amount of between 300 mM and500 mM NaCl, preferably 350 to 450 mM NaCl, and more preferably about400 mM NaCl.

In addition, buffers are present in these formulations, because it isbelieved that the Factor VIII molecule can be adversely affected by pHshifts during lyophilization. The pH should preferably be maintained inthe range of between 6 and 8 during lyophilization, and more preferablyat a pH of about 7. The buffering agent can be any physiologicallyacceptable chemical entity or combination of chemical entities whichhave the capacity to act as buffers, including histidine, Tris, BIS-TrisPropane, PIPES, MOPS, HEPES, MES and ACES. The full chemicaldesignations of these buffering agents is listed in Table 1 below.Typically, the buffering agent is included in a concentration of 10-50mM. When histidine is added to the formulations, concentrations of over20 mM and preferably about 25 mM are used, alone or in combination withother buffers such as Tris. Histidine is especially preferred for use inthe compositions of the present invention, as described in greaterdetail below.

TABLE 1 Buffering Agents Tris tris-(hydroxymethyl)-aminomethane BIS-TrisPropane 1,3-bis-[tris-(hydroxy-methyl)methylamino]-propane PIPESpiperazine-N,N′-bis-(2-ethanesulfonic acid) MOPS 3-{N-morpholino)propanesulfonic acid HEPESN-2-hydroxyethyl-piperazine-N′-2-ethanesulfonic acid MES2-(N-morpholino) ethanesulfonic acid ACESN-2-acetamido-2-aminoethanesulfonic acid

In order to preserve the activity of Factor VIII, it is important thatthe formulations of the present invention also include calcium oranother divalent cation able to interact with Factor VIII and maintainits activity, presumably by maintaining the association of the heavy andlight chains of Factor VIII. Between 1 mM and 5 mM of a calcium salt canbe used, more preferably 3-4 mM, and most preferably about 4 mM. Thecalcium salt is preferably calcium chloride, but can also be othercalcium salts such as calcium gluconate, calcium glubionate, or calciumgluceptate.

The Factor VIII compositions of the present invention also preferablyinclude a surfactant, preferably in an amount of 0.1% or less, and morepreferably in an amount of about 0.03%. The surfactant can, for example,be chosen from the group consisting of polysorbate 20, polysorbate 80,pluronic polyols, and Brij 35 (polyoxyethylene 23 lauryl ether). Severalgrades of pluronic polyols (sold under the trade name Pluronic,manufactured by the BASF Wyandotte Corporation) are available. Thesepolyols, of diversified molecular weight (from 1,000 to over 16,000) andphysicochemical properties have been used as surfactants. Pluronic F-38,of a molecular weight of 5,000 and Pluronic F-68, molecular weight9,000, both contain (by weight) 80 per cent hydrophilic polyoxyethylenegroups and 20 percent hydrophobic polyoxypropylene groups. Tween-80, acommercial polysorbate, however, is preferred in the presentformulations, in particular vegetable-derived Tween-80.

The Factor VIII formulations of the present invention also preferablyinclude an antioxidant. The addition of antioxidants to the lyophilizedformulations of the invention has been found to improve the stability ofthese formulations, and thus extend their shelf lives. The antioxidantsused must be compatible for use with a pharmaceutical preparation, andin addition are preferably water soluble. When adding antioxidants to aformulation, it is preferable to add such antioxidants as late in theprocess prior to lyophilization as possible, in order to avoidspontaneous oxidation of the antioxidant. Table 2 below lists suitableantioxidants, which are available commercially through companies such asCalbiochem and Sigma.

TABLE 2 Antioxidants N-Acetyl-L-Cysteine/Homocysteine Glutathione6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) Lipoicacid Methionine Sodium Thiosulfate Platinum Glycine-glycine-histidine(tripeptide) Butylatedhydroxytoluene (BHT)

Of the foregoing antioxidants, glutathione is preferred. Concentrationsin the range of about 0.05 mg/ml to more than 1.0 mg/ml have all beenfound to enhance the stability of Factor VIII compositions, and it isbelieved that higher concentrations would also be useful (up to thepoint of any toxic effects or adverse manufacturing effects, such as adepression of the glass transition temperature of the lyophilizedproduct).

It has been found in particular that the combination of histidine andglutathione produces synergistically beneficial effects on the stabilityof Factor VIII compositions. Histidine, while acting as a buffer, canalso act as a metal chelator. To the extent that Factor VIIIinactivation is caused by metal-induced oxidation, histidine cantherefore act to stabilize Factor VIII by binding such oxidizing metalions. It is believed that by binding these metals, the glutathione (orindeed any other antioxidant present) is thereby able to provide furtherantioxidative protection, since the oxidative effect of the metal ionsbound by the histidine has been contained.

Other chelating agents might also be used in the compositions of thepresent invention. Such agents should preferably bind metals such ascopper and iron with greater affinity than calcium, if a calcium salt isbeing used in the composition. One such chelator is deferoxamine, achelating agent that facilitates the removal of Al++ and iron.Deferoxamine Mesylate, C25H48N6O8*CH4O3S, can be obtained from Sigma(Sigma Prod. No. D9533). It is an aluminum and iron(II) chelator whichchelates iron (as a 1:1 chelate complex) only in the +3 oxidation state,not +2 oxidation state, and can also bind manganese ion and othermetals. Deferoxamine can be used advantageously in an amount of 0.25mg/l.

The Factor VIII used in the present formulations can be either highlypurified human plasma-derived Factor VIII or more preferably can berecombinantly produced Factor VIII. Recombinant Factor VIII can beproduced by Chinese hamster ovary (CHO) cells transfected with a vectorcarrying a DNA sequence coding for the Factor VIII molecule. Methods forcreating such transfected CHO cells are described, inter alia, in U.S.Pat. No. 4,757,006 to Toole, Jr., though alternative methods are alsoknown to the art (see, e.g., U.S. Pat. No. 4,868,112, also to Toole,Jr., and PCT International Application WO-A-91/09122). The methods usedto culture such CHO cells to produce Factor VIII are also known to theart, for example in European Patent Application No. 0 362 218 toGenetics Institute, entitled “Improved method for producing FactorVIII:C-type proteins.” Recombinant Factor VIII can, however, also beproduced in other cell lines, such as baby hamster kidney (BHK) cells.The Factor VIII molecule itself, if recombinantly produced, can beeither full-length Factor VIII or a deletion derivative thereof, such asa B domain-deleted Factor VIII molecule.

While the Factor VIII compositions described in this application can belyophilized and reconstituted in the indicated concentrations, one ofskill in the art will understand that these preparations can also bereconstituted in more dilute form. For example, a preparation accordingthe present invention which is lyophilized and/or normally reconstitutedin 2 ml of solution can also be reconstituted in a larger volume ofdiluent, such as 5 ml. This is particularly appropriate when the FactorVIII preparation is being injected into a patient immediately, since inthis case the Factor VIII is less likely to lose activity, which mayoccur more rapidly in more dilute solutions of Factor VIII.

Formulation and Lyophilization Development

In order to achieve maximal stability, the Factor VIII compositions ofthe present invention are preferably lyophilized. During lyophilization,Factor VIII is converted from being in an aqueous phase to being in anamorphous solid phase, which is thought to protect the protein fromchemical and/or conformational instability. The lyophilized preparationnot only contains an amorphous phase, but also includes a componentwhich crystallizes during lyophilization. This is thought to allow therapid lyophilization of the Factor VIII composition and the formation ofa more elegant cake (that is, a cake with minimal shrinkage from thesides of the container in which it was lyophilized). In the formulationsof the present invention, the stabilizing agents have been selected toexist primarily in an amorphous phase of the lyophilized product, whilethe bulking agents (except HES) have been selected to crystallize duringfreezing.

Both the Factor VIII and the stabilizer are preferably dispersed in theamorphous phase of the lyophilized cake. The mass of the stabilizer isalso preferably large compared to the other excipients in the amorphousform. In addition, the apparent glass transition temperature (T_(g)′) ofthe amorphous phase is preferably relatively high during freeze-drying,and the glass transition temperature (Tg) of the solid is likewisepreferably high during storage. Crystallization of sodium chloride inthe product was found to be desirable, since amorphous sodium chloridewill depress the T_(g)′ of the amorphous phase.

In order to avoid the collapse of the cake of a particular composition,primary drying is preferably carried out at a product temperature belowthe apparent glass transition temperature of the freeze concentrate. Anincrease in drying time may also be required to offset a decrease inT_(g). Further information on lyophilization may be found in Carpenter,J. F. and Chang, B. S., Lyophilization of Protein Pharmaceuticals,Biotechnology and Biopharmaceutical Manufacturing, Processing andPreservation, K. E. Avis and V. L. Wu, eds. (Buffalo Grove, Ill.:Interpharm Press, Inc.), pp. 199-264 (1996).

Example 1

The effects of the concentration of Factor VIII and of the addition of astabilizer on the recovery of Factor VIII were investigated in severalstudies. These studies were performed using mannitol as a model bulkingagent and sucrose as a model stabilizer. The three sample formulationsdescribed in Table 3 below were used in these studies. All formulationsused in these studies included 10 mM Tris, 200 mM NaCl, 8% mannitol, 4mM CaCl₂, and 0.02% Tween-80 and were conducted at pH 7.0.

TABLE 3 Sample Initial Factor VIII I.D. (IU/ml) Sucrose % IA 600 — IB 60— IC 60 2

These samples were lyophilized using the freeze-drying cycle shown inTable 4 below in order to maintain a product temperature below theapparent glass transition temperature (T_(g)′). Differential scanningcalorimetric (DSC) studies indicated the presence of a transition atapproximately −40° C. in the mannitol formulations. In order to maintaina product temperature below this value, the shelf temperature was set to−32° C. during primary drying. Primary drying under these conditions wasperformed for about 55 hours, with a total cycle time of about 80 hours.

TABLE 4 Freezing/Processing Method Description I Cool to +5° C.;(Freezing) Cool to −5° C. at 1° C./minute, hold for 20 minutes; Cool to−20 ± 5° C. at 1° C./minute, hold for 1 hour (up to 3 hours); Cool to−45° C. at 0.5° C./minute, hold for 1 hour. II Freeze per method I Holdat −35° C. for 48 hours. III Freeze per method I Hold at −35° C. for 48hours; Hold at −20° C. for 48 hours. IV Shelf −32° C. during primarydrying for (Freeze-drying) about 55 hours (up to 100 hours); Product <−40° C. during primary drying; Ramp from −32° C. to +40° C. at 0.2°C./minute; Shelf +40° C. during secondary drying for 3 hours.

The Factor VIII activity of these samples, as determined by theone-stage clotting assay, was compared against a control held at −45° C.The assay results are shown in Table 5 below.

TABLE 5 % Loss in Factor VIII Activity During Each Step Formulation ICProcessing Formulation IA Formulation IB (60 IU/ml, 2% Method (600IU/ml) (60 IU/ml) Sucrose) I 6.7 37.5 41.7 II 2.0 9.3 3.9 III 7.3 11.65.0 IV 20.0 24.2 18.3 (Lyophilization)

These results indicate that protein concentration has an effect on therecovery of Factor VIII during freezing. Formulations containing 60IU/ml lost approximately 37%-42% of the initial Factor VIII activityduring the freezing step, while 6.7% of Factor VIII activity was lostfor the formulation containing 600 IU/ml. These results indicate that ahigher protein concentration has a protective effect during freezing.Although sucrose provided some protection to the Factor VIII during theintermediate temperature holds as well as during freeze-drying, itfailed to protect the protein during the initial freezing step.

Example 2

Following the development of the lyophilization process outlined inExample 1, further optimization of this process was undertaken. It hasbeen found that a lyophilized composition having a higher glasstransition temperature, (and, theoretically, better Factor VIIIstability) can be produced by: (1) lowering the freezing temperatureinitially to −45° C. or lower (such as down to about −50° C. or −55°C.); (2) raising the temperature to −20° C. or −22° C. (+5° C.); andthen (3) lowering the temperature again to −45° C. or lower. Thetemperature is lowered or raised, as the case may be, at a rate ofbetween about 0.5° C. and about 1.0° C. per minute. Once the desiredtemperature is reached, the composition is held at that temperature forbetween 1 and 3 hours. This improved freezing cycle is shown in Table 6below.

TABLE 6 Freezing Method Description I Cool to +5° C.; Cool to −5° C. at0.5-1° C./minute, hold for 20 minutes; Cool to between−55° C. and −45°C. at 0.5-1° C./ minute, hold for about 1 hour; Warm to −22° C. (±5° C.)at 0.5-1° C./minute, hold for 1 to 3 hours; Cool to −45° C. at 0.5-1°C./minute, hold for about 1 hour.

Unless otherwise indicated, the temperatures referred to in this exampleand in other examples refer to the shelf temperature of the lyophilizerand not to the temperature of the product per se. Following the improvedfreezing cycle, the remainder of the lyophilization process can beconducted as outlined in Example 1 above, or otherwise as describedfurther herein or as determined by one of skill in the art.

This improved lyophilization process was found to be useful forformulations which include glycine as the bulking agent as well as thosewhich use mannitol. It is further believed to have applicability toformulations which make use of the other bulking agents of the presentinvention as well.

Example 3

It is believed that in order to produce a freeze-dried product withacceptable cake appearance and glass transition temperature, the bulkingagent of lyophilized pharmaceutical preparations which contain sodiumchloride, such as glycine or mannitol, may need to be crystallized. Thefollowing improved lyophilization process for crystallizable bulkingagents was therefore developed.

TABLE 7a Freezing Steps Process Step Temperature Duration of StepInitial freezing −40° C. or less 1 hour First annealing between −23° C.and −27° C. 3 hours Second freezing −55° C. 1 hour Second annealing −36°C. 4 hours Third freezing −50° C. 1 hour

TABLE 7b Freeze-Drying Steps Process Step Temperature Duration of StepPrimary Drying −35° C.   up to 100 hours Secondary Drying: First step40° C. 3 hours Secondary Drying: Second step 45° C. 3 hours SecondaryDrying: Third step 50° C. 3 hours

In the freezing steps, changes in the temperatures occurred at a rate ofbetween about 0.5° C./minute and 1° C./minute. It is believed that stepsof longer duration would also be effective.

Prior to the first freezing step, the temperature is brought to betweenabout 2° C. and 8° C. for about one hour for the purpose of bringing allthe vials to approximately the same temperature. After this thelyophilizer is cooled to −5° C. The first freezing step should beperformed at a temperature less than −30° C., preferably below −35° C.,and more preferably at about −40° C. Following this, the first annealingstep should occur at a temperature of between −30° C. and −19° C., morepreferably either between about −25° C. and −28° C. (if glycine is thebulking agent) or between −21° C. and −24° C. (if mannitol is thebulking agent), with the temperatures of −23° C. and −26° C. being mostpreferred, at which temperatures it is believed that the crystallizablebulking agents crystallize, at least in part. However, the lower rangearound −27° C. is not recommended for formulations containing mannitoland arginine. This step is preferably carried out for about 3 hours.

Following the first annealing step, the temperature is lowered,preferably to less than about −50° C. and more preferably to less than−55° C., for about 1 hour. It is believed that the sodium chloride inthe preparation nucleates at this time.

During the second annealing step, the temperature of the pharmaceuticalpreparation is raised to between about −30° C. and −39° C., andpreferably to about −33° C. for mannitol-containing compositions and−36° C. for glycine-containing compositions. It is believed that NaClcrystal growth occurs at this time, at least in part. This step ispreferably conducted for about 4 hours. Following this, the temperatureof the lyophilizer is reduced to about −50° C., preferably for about 1hour in order to reduce the temperature of the preparation.

In the freeze-drying steps which follow, changes in temperature occurredat a rate of between about 0.1° C./minute and 0.5° C./minute. Afterreducing the pressure in the lyophilizer to about 65 mTorr, thetemperature is raised to between about −32° C. and −35° C. for primarydrying. Ice crystals in the preparation will sublimate at thistemperature. This step is performed for up to about 100 hours, or untilmost of the ice has been sublimated from the preparation. The point atwhich most of the ice has sublimated can be determined, for example,using a dewpoint sensor, which indicates the end of the sublimation ofice when the readings decrease (the point of inflection).

Following primary drying, the temperature is raised to +40° C.,preferably at a rate of 0.2° C./minute, to initiate secondary drying toremove further water from the preparation. This temperature ispreferably maintained for about three hours. Second and third secondarydrying steps follow this first step, where the temperature is raised toabout +45° C. for about three hours and then to about +50° C. for threemore hours in order to reduce the moisture in the lyophilized cake toless than 2% (w/w).

Example 4

Further studies were performed to examine specifically the effect ofhistidine on lyophilized Factor VIII compositions containing glycine ormannitol as bulking agents. Non-reversing heat flow (Modulated DSC,mDSC) was used to detect the crystallization of these bulking agentsduring cooling. Both the temperature of crystallization and the totalheat of crystallization were determined from the crystallizationexotherm. The appearance of the NaCl eutectic melt endotherm duringwarming was used to detect NaCl crystallization. In mDSC, the extent ofcrystallization was determined as the ratio of the enthalpy of meltingof the formulation to the enthalpy of melting of pure NaCl solution byusing the total heat flow signal. In addition, X-ray diffractionanalyses were performed in order to determine the extent ofcrystallization in the lyophilized formulations.

While histidine concentrations less than 20 mM did not significantlyimpact the crystallization of glycine, 50 mM histidine reduced theextent of glycine crystallization. Well-defined NaCl crystallizationexotherms were not observed during cooling of formulations containingglycine. However, eutectic melting endotherms during heating indicatedthat NaCl was crystallized (>50%) after cooling lower than −50° C. andannealing at −30° C., −35° C. and −40° C. The inclusion of 50 mMhistidine in the glycine-containing formulation retarded NaClcrystallization. Consequently, the annealing time was increased 3-foldfor such formulations in order to achieve an equivalent crystallinity.

However, the effect of 20 mM histidine on the crystallization of NaCl inthe glycine-containing formulations was minimal. In freeze-dryingstudies, collapse of the lyophilized cake was observed visually inglycine-containing formulations containing 50 mM histidine. X-ray powderdiffraction data indicated a decrease in the crystallinity of NaCl insamples containing histidine. In mannitol-containing formulations,typically 83%-90% of the sodium chloride crystallized during coolingbetween −40° C. and −50° C. without the need for annealing. Whileinclusion of 20 mM histidine to the formulation suppressed NaClcrystallization during cooling, annealing resulted in approximately 40%crystallization of the NaCl.

Therefore, in formulations containing a crystallizable bulking agent,such as glycine or mannitol, and NaCl, the inclusion of histidine maydecrease the extent of crystallization of NaCl. Although this could insome cases lead to the collapse of the cake which is formed duringlyophilization, the use of relatively lower concentrations of histidinein such formulations can mitigate this effect. Nonetheless, acceptablecakes have been formed with concentrations of histidine of 35 mM and 50mM. Histidine may also be preferable to HEPES as a buffer in mannitol-and glycine-based formulations, as the use of HEPES has been observed tolower the Tg′ to a greater extent than a similar amount of histidine.

Example 5

The physical characteristics of a number of potential Factor VIIIformulations, including seven candidate stabilizers and five bulkingagents, were evaluated in another study. In addition to a bulking agentand stabilizer, all formulations listed in Table 8 below (except forformulation 11) contained 10 mM TrisHCl, 200 mM NaCl, 0.02% Tween-80, 4mM CaCl₂ and were at pH 7.0. Formulation 11 contained 10 mM TrisHCl,0.02% Tween-80, and 4 mM CaCl₂, also at pH 7.0. All pH measurements wereperformed at ambient temperature.

TABLE 8 Sample Protein I.D. Bulking Agent Stabilizer 1 8% Mannitol 2%Sucrose 2 8% Mannitol 2% Trehalose 3 8% Mannitol 2% Raffinose 4 8%Mannitol 2% Arginine 5 8% Mannitol 2% Lysine 6 8% Mannitol 2% Sorbitol 78% Mannitol 2% Glycerol 8 4% Hydroxyethyl 2% Sucrose Starch 9 8% Glycine2% Sucrose 10 8% Glycine 2% Trehalose 11 400 mM NaCl 2% Sucrose 12 8%Alanine 2% Sucrose

Collapse temperature measurements by freeze-dry microscopy and thermaltransition measurements by DSC were used to predict freeze-dryingbehavior. DSC, X-ray powder diffraction and polarized light microscopywere also used to determine the crystallinity of the lyophilizedsamples. The reconstitution time and the appearance of the samples werealso evaluated. The results of all of these measurements are summarizedin Table 9 below.

TABLE 9 Recon- Water Sample T_(pc) T_(c) T_(g) stitution Content I.D. (°C.) (° C.) (° C.) (seconds) (%) Appearance 1 −14 −10 54 64 n/c Elegant 2−20 −15 53 62 1.4 Top partially collapsed 3 −15 −10 54 77 1.7 Elegant 4— — — — — Partial collapse 5 — — — — — Collapsed 6 n/c n/c <10° C.* 630.6 Elegant 7 — — <10° C.* — — Elegant 8 — — 86 49 0.7 Elegant butshrunk from sides 9 — — 54 22 0.8 Elegant 10 — — 63 18 — Elegant 11 — —66 11 0.4 Elegant (layer on bottom) 12 — — — 57 0.5 Elegant *Sorbitoland Glycerol have glass transitions at <10° C. The DSC scan range didnot include temperatures in this range. n/c = not clear T_(pc) =Temperature at which partial collapse occurs in the freeze-drymicroscope T_(c) = Temperature at which total collapse occurs in thefreeze-dry microscope T_(g) = Glass transition temperature

With the exception of mannitol:lysine, all of the formulations appearedto have adequate physical appearance. Lysine interfered with thecrystallization of both mannitol and glycine, which caused a depressionin the glass transition temperature and a collapse of the lyophilizedcake.

Example 6

The Factor VIII compositions described in Table 8 above were placed instorage at −70° C., 25° C., 40° C., and 50° C. for varying lengths oftime in order to evaluate their stability. Factor VIII activity levelswere evaluated after 2 weeks, 1 month, 2 months, and 3 months, and theresults are summarized in Table 10 below. Two of the samples, oneemploying mannitol as the bulking agent and sorbitol as the stabilizer,and the other employing mannitol as the bulking agent and glycerol asthe stabilizer, exhibited poor stability. The remaining formulations allexhibited the ability to stabilize Factor VIII.

TABLE 10 Temperature % of initial at month Formulation Description (°C.) 0 0.5 1 2 3 Glycine:Sucrose −70 100.00 97.43 101.71 99.89 97.97 25100.00 85.44 40 100.00 79.87 71.52 63.06 50 100.00 76.34 67.99 52.1447.64 Glycine:Trehalose −70 100.00 89.22 96.00 95.90 94.64 25 100.0083.17 40 100.00 79.93 72.42 68.03 50 100.00 80.97 64.28 57.60 50.92Mannitol:Trehalose −70 100.00 91.32 97.72 96.10 98.26 25 100.00 85.79 40100.00 82.54 70.72 59.44 50 100.00 66.16 65.51 48.81 52.06Mannitol:Sucrose −70 100.00 100.45 100.56 105.47 99.22 25 100.00 87.0440 100.00 85.59 80.78 55.42 50 100.00 81.68 75.53 57.88 43.46Mannitol:Arginine −70 100.00 102.26 105.53 103.72 105.08 25 100.00 95.1540 100.00 91.53 80.93 69.19 50 100.00 82.28 68.06 56.32 45.94Mannitol:Raffinose −70 100.00 93.88 98.41 100.68 103.62 25 100.00 83.1340 100.00 81.09 73.61 67.16 50 100.00 71.69 68.52 54.25 47.11Mannitol:Glycerol −70 25 40 50 Mannitol:Sorbitol −70 100.00 104.06 25100.00 40 100.00 50 100.00 32.73 HES:Sucrose −70 100.00 102.74 103.03100.90 25 100.00 40 100.00 76.89 77.47 50 100.00 71.47 67.40 30.02NaCl:Sucrose −70 100.00 88.54 88.44 95.58 25 100.00 40 100.00 71.5658.30 50 100.00 52.71 37.90 30.34 Alanine:Sucrose −70 100.00 109.78109.67 108.96 25 100.00 40 100.00 92.99 73.03 50 100.00 83.25 74.9157.65 Glycine:Raffinose −70 100.00 111.57 114.51 105.25 25 100.00 40100.00 89.20 82.10 50 100.00 93.21 72.22 53.24

Example 7

Based on the information developed during the studies described inExamples 5 and 6, it was decided that candidate formulations having theexcipients shown in Table 11 below would be further developed.

TABLE 11 Excipient Concentration mannitol or glycine 6-9% arginine ortrehalose 1-3% tween 80 0.005-0.04%  NaCl 200-250 mM CaCl2 3-5 mM TRIS20-30 mM histidine or HEPES 10-50 mM glutathione 0.15-0.25 mg/ml

Based on these parameters, the following specific formulations weredeveloped:

TABLE 12 Formulation #1 Formulation #2 Formulation #3 10 mM HEPES 10 mMHEPES 10 mM HEPES 20 mM Tris 20 mM Tris 20 mM Tris 225 mM NaCl 225 mMNaCl 225 mM NaCl 0.03% (v/v) Tween-80 0.03% (v/v) Tween-80 0.03% (v/v)Tween-80 8% (w/v) mannitol 8% (w/v) glycine 8% (w/v) mannitol 2% (w/v)trehalose 2% (w/v) trehalose 2% (w/v) arginine 0.2 mg/ml reduced 0.2mg/ml reduced 0.2 mg/ml reduced glutathione glutathione glutathione 4 mMCaCl₂ 4 mM CaCl₂ 4 mM CaCl₂ Formulation #4 Formulation #5 25 mMhistidine 25 mM histidine 20 mM Tris 20 mM Tris 225 mM NaCl 225 mM NaCl0.03% (v/v) Tween-80 0.03% (v/v) Tween-80 8% (w/v) mannitol 8% (w/v)glycine 2% (w/v) trehalose 2% (w/v) trehalose 0.2 mg/ml reduced 0.2mg/ml reduced glutathione glutathione 4 mM CaCl₂ 4 mM CaCl₂

1. A Factor VIII composition formulated without adding albumin to saidcomposition and comprising the following formulation excipients inaddition to Factor VIII: 4% (w/v) to 10% (w/v) of a bulking agent,wherein said bulking agent is mannitol; 1% (w/v) to 4% (w/v) of astabilizing agent selected from the group consisting of sucrose,trehalose, and raffinose; 1 mM to 5 mM calcium salt; 100 mM to 300 mMNaCl; and a buffering agent for maintaining a pH of approximatelybetween 6 and
 8. 2. The Factor VIII composition of claim 1, wherein saidbulking agent is present in an amount of about 8% (w/v).
 3. The FactorVIII composition of claim 1, wherein said stabilizing agent is presentin an amount of about 2% (w/v).
 4. The Factor VIII composition of claim1, wherein said stabilizing agent is sucrose.
 5. The Factor VIIIcomposition of claim 1, wherein said stabilizing agent is trehalose. 6.The Factor VIII composition of claim 1, wherein said calcium salt iscalcium chloride.
 7. The Factor VIII composition of claim 1, whereinsaid NaCl is present in an amount of from 150 mM to 250 mM.
 8. TheFactor VIII composition of claim 1, wherein said NaCl is present in anamount of about 225 mM.
 9. The Factor VIII composition of claim 1,wherein said calcium salt is present in an amount of from 1 mM to 5 mM.10. The Factor VIII composition of claim 1, wherein said composition isin lyophilized form.
 11. The Factor VIII composition of claim 1, whereinsaid buffering agent comprises between about 10 mM and about 50 mMhistidine.
 12. The Factor VIII composition of claim 11, wherein thehistidine is present in an amount of about 25 mM.
 13. The Factor VIIIcomposition of claim 1, wherein said buffering agent comprises a memberselected from the group consisting of tris-(hydroxymethyl)-aminomethane,1,3-bis-[tris-(hydroxy-methyl)methylamino]-propane,piperazine-N,N′-bis-(2-ethanesulfonic acid), 3-(N-morpholino)propanesulfonic acid, N-2-hydroxyethyl-piperazine-N′-2-ethanesulthnicacid, 2-(N-morpholino) ethane-sulfonic acid andN-2-acetamido-2-aminoethanesulfonic acid.
 14. The Factor VIIIcomposition of claim 11, wherein said buffering agent further comprisesa member selected from the group consisting oftris(hydroxymethyl)-aminomethane,1,3-bis-[tris-(hydroxy-methyl)methylamino]-propane,piperazine-N,N′-bis-(2-ethanesulfonic acid), 3-(N-morpholino)propanesulfonic acid, N-2-hydroxyethyl-piperazine-N′-2-ethanesulfonicacid, 2-(N-morpholino) ethane-sulfonic acid andN-2-acetamido-2-aminoethanesulfonic acid.
 15. The Factor VIIIcomposition of claim 14, wherein said buffering agent further comprisestris-(hydroxymethyl)-aminomethane.
 16. The Factor VIII composition ofclaim 15, wherein the tris-(hydroxymethyl)-aminomethane is present in anamount of about 20 mM.
 17. The Factor VIII composition of claim 1,further comprising a surfactant.
 18. The Factor VIII composition ofclaim 17, Wherein the surfactant is present in an amount of about 0.03%(w/v).
 19. The Factor VIII composition of claim 17, wherein saidsurfactant is selected from the group consisting of polyoxyethylene (20)sorbitan fatty acid ester, polyoxyethylene (80) sorbitan fatty acidester, polyoxyalkylene ether, and polyoxyethylene 35 lauryl ether. 20.The Factor VIII composition of claim 19, where said surfactant ispolyoxyethylene (80) sorbitan fatty acid ester and is present in anamount of between 0.02% (w/v) and 0.1% (w/v).
 21. The Factor VIIIcomposition of claim 1, further comprising an antioxidant.
 22. TheFactor VIII composition of claim 21, wherein said antioxidant isglutathione.
 23. The Factor VII composition of claim 22, wherein saidglutathione is present in an amount of between 0.05 mg/ml and 1.0 mg/ml.24. The Factor VIII composition of claim 1 in combination with adiluent.
 25. The Factor VIII composition of claim 1 in combination with2 ml of diluent.
 26. The Factor VIII composition of claim 1 incombination with 5 ml of diluent.
 27. A Factor VIII compositionformulated without adding albumin to said composition and comprising thefollowing formulation excipients in addition to Factor VIII: 2% (w/v) to6% (w/v) hydroxyethyl starch; 1% (w/v) to 4% (w/v) of a stabilizingagent selected from the group consisting of sucrose, trehalose, andraffinose; 1 mM to 5 mM calcium salt; 100 mM to 300 mM NaCl; and abuffering agent for maintaining a pH of approximately between 6 and 8,wherein said buffering agent comprises between about 10 mM and about 50mM histidine.
 28. The Factor VIII composition of claim 27, wherein saidhydroxyethyl starch is present in an amount of about 4%.
 29. The FactorVIII composition of claim 27, wherein said NaCl is present in an amountof about 200 mM.
 30. The Factor VIII composition of claim 27, whereinsaid stabilizing agent is present in an amount of about 2% (w/v).
 31. Animproved method of lyophilizing an aqueous pharmaceutical formulationcomprising a crystallizable bulking agent, a stabilizing agent, acalcium salt, and NaCl, wherein said method comprises the steps of: (a)freezing the aqueous pharmaceutical formulation at a temperature of lessthan about 35° C., (b) annealing the pharmaceutical formulation atbetween about −30° C. and −19° C.; (c) lowering the temperature of thepharmaceutical formulation to between −45° C. and 55° C.; (d) annealingthe pharmaceutical formulation at between about −30° C. and −39° C.; and(e) freeze-drying the pharmaceutical formulation.
 32. The method ofclaim 31, wherein the temperature in step (c) is less than −50° C. 33.The method of claim 31, wherein said pharmaceutical formulation furthercomprises Factor VIII.
 34. The method of claim 31, wherein saidpharmaceutical formulation further comprises a buffering agent.
 35. Themethod of claim 34, wherein said buffering agent comprises histidine.36. The method of claim 34, wherein said buffering agent comprisestris-(hydroxymethyl)-aminomethane.
 37. The method of claim 31, whereinsaid bulking agent is mannitol.
 38. The method of claim 31, wherein saidstabilizing agent is trehalose.
 39. The method of claim 31, wherein saidcalcium salt is calcium chloride.